№ 3 / 2016 / 13-22

Chemical and pharmacological properties of subtilisins

Author: Madonov P.G.¹, Mishenina S.V.², Kinsht D.N.³, Kikhtenko N.V.⁴

Abstract

Subtilisins are a class of hydrolases proteolytic enzymes produced by the bacteria Bacillus subtilis. The study of the chemical and pharmacological properties of subtilisin has been actively conducted the last 20 years. The interest has been caused by the presence of subtilisin fibrinolytic, thrombolytic and anticoagulation action. In this case, separated subtilisins show different fibrinolytic activity, and some of them are even more active than urokinase. Subtilisins effect does not have site-specificity and is directed to those proteins that have lost native globular structure (e.g., fibrinogen, into fibrin turned). It is known that the population of Japan, China and North East India are characterized by the lowest risk of heart diseases and dementia, perhaps this is due to the high content of fibrinolytic enzymes in traditional food (nattokinase from soybeans). Despite the large number of experimental studies, the subtilisins practical use in medicine remains limited. The only registered drug for thrombosis treatment is Russian medicine Trombovazim® created in 2009 and based on immobilized subtilisin.

Key words

proteolytic enzymes, subtilisins, serine proteases, fibrinolytic activity, anticoagulant potential

References

Balaban N.P., Sharipova M.R. Practical application of bacillary protease. // Scientists notes of the Kazan University. Series: Science. 2011. 153. (2). 29-40.

Balaban N.P., Sharipova M.R., Itskovich E.L. et al. Secreted serine protease from the spore forming bacterium Bacillus intermedius 3-19 // Biochemistry (Moscow). 1994. 59. (9). 1093-1099.

Vereschagin E.I., Kinsht D.N., Madonov P.G. et al. The clinical efficacy of a new national thrombolytic drug Trombovazim. // Man and medicine: a collection of materials VII Russian Congress. M., 2009. 629.

Vereschagin E.I., Kinsht D.N., Madonov P.G. et al. Pharmacokinetics, antithrombotic and thrombolytic effect of a new national oral drug Trombovazim. // Man and medicine: a collection of materials VII Russian Congress. M., 2009. 629-630.

Danilova J.V., Cheremin A.M., Zamaleeva A.I. et al. Thrombolytic and fibrinolytic activity of bacterial proteases. // Cellular Transplantation and Tissue Engineering. 2012. 7. (3). 49-51.

Itskovich E.L., Lutova L.I., Balaban N.P. et al. Thrombolytic and anticoagulant properties of thiol- dependent serine proteinase bacillus intermedius 3-19. // Problems of Medical Chemistry. 1998. 3. 288-291.

Kinsht D.N., Vereschagin E.I., Madonov P.G. et al. Efficacy Trombovazim drug in the treatment of chronic venous insufficiency. // Man and medicine: a collection of materials VII Russian Congress. M., 2009. 127-128.

Lutova L.V., Andreenko G.V., Karabasova M.A. et al. Research thrombolytic properties of thiol dependent serine proteinase (TSP) from Thermoactinomyces vulgaris in vivo. // J. Appl. biochemical and microbiology. 1990. 26. (5). 623-628.

Plotnikov M.B., Dygai A.M., Aliev O.I. et al. Antithrombotic and thrombolytic effects of a new proteolytic preparation Thrombovazim (Russia). // Bulletin of Experimental Biology and Medicine. 2009. 147. (4). 418-421.

Rudenskaya G.N., Lutova L.V., Andreenko G.V. et al. The study of fibrinolytic and thrombolytic properties of thiol dependent serine proteinase Thermoactinomyces vulgaris in vitro. // J. Appl. biochemical and microbiology. 1987. 23. (6). 754-760.

Trofimov A.O., Kravets L.Y. On the question of fibrinolysis in intracranial hemorrhage. // V Congress of Neurosurgeons of Russia: Congress materials. Ed. Safin Sh.M. Ufa.: Bashkortostan Health. 2009. 464.

Agrebi R., Haddar A., Hmidet N. et al. SF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26: Purification, biochemical and molecular characterization. // Process Biochemistry. 2009. 44. (11). 1252-1259.

Bi Q., Han B., Feng Y. et al. Antithrombotic effects of a newly purified fibrinolytic protease from Urechis unicinctus. // Thrombosis Research. 2013. 132. (2). e135-e144.

Choi D.B., Cha W.-S., Park N. et al. Purification and characterization of a novel fibrinolytic enzyme from fruiting bodies of Korean Cordyceps militaris // Bioresource Technology. 2011. 102. (3). 3279-3285.

Fujita M., Nomura K., Hong K. et al. Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto, a popular soybean fermented food in Japan // Biochemical and Biophysical Research Communications. 1993. 197. (3) 1340-1347.

Kim D.-W., Sapkota K., Choi J.-H. et al. Direct acting anti-thrombotic serine protease from brown seaweed Costaria costata. // Process Biochemistry. 2013. 48. (2). 340-350.

Kim S.B., Lee D.W., Cheigh C.I. et al. Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybean, Tempeh // J. Ind. Microbiol. Biotechnol. 2006. 33. (6). 436-444.

Kim W., Choi K., Kim Y. et al. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from chungkook-jang // Appl. Environ. Microbial. 1996. 62. (7). 2482-2488.

Ko J.H., Yan J.P., Zhu L. et al. Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02 // Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2004. 137. (1). 65-74.

Kotb E. Activity assessment of microbial fibrinolytic enzymes. // Appl Microbiol Biotechnol. 2013. 97. (15). 6647-6665.4

Mahajan P.M., Nayak S., Lele S.S. Fibrinolytic enzyme from newly isolated marine bacterium Bacillus subtilis ICTF-1: Media optimization, purification and characterization. // Journal of Bioscience and Bioengineering. 2012. 113. (3). 307-314.

Majumdar S., Sarmah B., Gogoi D. et al. Characterization, mechanism of anticoagulant action, and assessment of therapeutic potential of a fibrinolytic serine protease (Brevithrombolase) purified from Brevibacillus brevis strain FF02B // Biochimie. 2014. 103. 50-60.

Mander P., Cho S.S., Simkhada J.R. et al. A low molecular weight chymotrypsin-like novel fibrinolytic enzyme from Streptomyces sp. CS624. // Process Biochemistry. 2011. 46. (7). 1449-1455.

Martinez D.E., Borniego M.L., Battchikova N. et al. SASP, a Senescence-Associated Subtilisin Protease, is involved in reproductive development and determination of silique number in Arabidopsis. // J Exp Bot. 2015. 66. (1). 161-174.

Montriwong A., Rodtong S., Yongsawatdigul J. Detergent-Stable Salt-Activated Proteinases from Virgibacillus halodenitrificans SK1-3-7 Isolated from Fish Sauce Fermentation. // Appl Biochem Biotechnol. 2015. 176. (2). 505-517.

Mukherjee A.K., Rai S.K., Thakur R. et al. Bafibrinase: A non-toxic, non-hemorrhagic, direct-acting fibrinolytic serine protease from Bacillus sp. strain AS-S20-I exhibits in vivo anticoagulant activity and thrombolytic potency. // Biochimie. 2012. 94. (6). 1300-1308.

Peng Y., Huang Q., Zhang R. et al. Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food. // Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 2003. 134. (1). 45-52.

Peng Y., Yang X., Zhang Y. Microbial fibrinolytic enzymes: an overview of source, production, properties, and thrombolytic activity in vivo. // Applied Microbiology and Biotechnology. 2005. 69. (2). 126-132.

Peng Y., Yang X.-J., Xiao L. et al. Cloning and expression of a fibrinolytic enzyme (subtilisin DFE) gene from Bacillus amyloliquefaciens DC-4 in Bacillus subtilis. // Research in Microbiology. 2004. 155. (3). 167-173.

Popovič T., Puizdar V., Brzin J. A novel subtilase from common bean leaves. // Febs Letters. 2002. 530. (1-3). 163-168.

Simkhada J.R., Cho S.S., Mander P. et al. Purification, biochemical properties and antithrombotic effect of a novel Streptomyces enzyme on carrageenan-induced mice tail thrombosis model. // Thrombosis Research. 2012. 129. (2). 176-182.

Singh T.A., Devi K.R., Ahmed G. et al. Microbial and endogenous origin of fibrinolytic activity in traditional fermented foods of Northeast India. // Food Research International. 2014. 55. 356-362.

Siritapetawee J., Thumanu K., Sojikul P. et al. A novel serine protease with human fibrino(geno)lytic activities from Artocarpus heterophyllus latex. // Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 2012. 1824. (7). 907-912.

Spreer A., Rüchel R., Reichard U. Characterization of an extracellular subtilisin protease of Rhizopus microsporus and evidence for its expression during invasive rhinoorbital mycosis. // Med Mycol. 2006. 44. (8). 723-731.

Sumi H., Hamada H., Tsushima H. et al. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet // Experientia 1987. 43. (10). 1110-1111.

Suzuki Y., Kondo K., Matsumoto Y. et al. Dietary supplementation of fermented soybean, natto, suppresses intimal thickening and modulates the lysis of mural thrombi after endothelial injury in rat femoral artery. // Life Sciences. 2003. 73. (10). 1289-1298.

Wang C., Ji B., Cao Y. et al. (Evaluating thrombolytic efficacy and thrombus targetability of RGDS-liposomes encapsulating subtilisin FS33 in vivo). // Xue Gong Cheng Xue Za Zhi. 2010. 27. (2). 332-336.

Wang C.T., Ji B.P., Li B. et al. Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi // J. Ind. Microbiol. Biotechnol. 2006. 33. (9). 750-758.

Wang S., Deng Z., Li Q. et al. A novel alkaline serine protease with fibrinolytic activity from the polychaete, Neanthes japonica // Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 2011. 159. (1). 18-25.

Wang S.-L., Wu Y.-Y., Liang T.-W. Purification and biochemical characterization of a nattokinase by conversion of shrimp shell with Bacillus subtilis TKU007. // New Biotechnology. 2011. 28. (2). 196-202.

Yan F., Yan J., Sun W. et al. Thrombolytic effect of subtilisin QK on carrageenan induced thrombosis model in mice. // J Thromb Thrombolysis. 2009. 28. (4). 444-448.

Yuan J., Yang J., Zhuang Z. et al. Thrombolytic effects of Douchi fibrinolytic enzyme from Bacillus subtilis LD-8547 in vitro and in vivo. // BMC Biotechnol. 2012. 12. 36-45.

About Authors (Correspondence):

Madonov P.G. – doctor of medical sciences, professor, head of the chair for pharmacology, clinical pharmacology and evidentiary medicine, e-mail: madonov@scpb.ru

Mishenina S.V. – candidate of medical sciences, associated professor of the chair for pharmacology, clinical pharmacology and evidentiary medicine

Kinsht D.N. – doctor of medical sciences, professor, head of the chair for pharmacology, clinical pharmacology and evidentiary medicine

Kikhtenko N.V. – researcher

Received: 20/07/2016